The present invention relates to polymers, in particular to polymers for the delivery of nucleic acid to a cell.
For many research applications in genetic manipulation and genetic engineering, it is necessary to express new or modified genes in living cells. However the uptake of DNA into cells is poor resulting in inconsistent expression. Similarly, gene therapy, antisense oligonucleotide therapy and gene vaccination require that DNA and DNA analogues can survive in a hostile biological environment, penetrate biological barriers, be taken up into cells and move to the correct subcellular compartment to exert their therapeutic effects.
The identification of defective genes responsible for disease states, either through the overproduction of key proteins, the production of defective proteins or the defective control of gene production, offers new possibilities for the treatment of disease. By controlling the defect at the genetic level a range of diseases could now be treated effectively rather than by merely treating the symptoms of these diseases. This has been achieved in some cases, or is believed to be achievable, by the expression of new competent genes, or by controlling the overproduction of unwanted gene products or by controlling the expression of genes. These processes could be achieved by the insertion of new DNA or by the administration and uptake of complementary strands of DNA or DNA analogues which inhibit the production or control the production of existing genes [1]. In both of these strategies it is necessary to deliver to the cell sufficient DNA to achieve modified cell expression. The DNA must also be delivered to the correct intracellular compartment to effect that change. While some DNA is taken up naturally into cells, the amount taken up is small and inconsistent, and expression of added DNA is poor. DNA is an inherently unstable material, particularly in a biological environment where many specific enzymes capable of degrading DNA are found [2]. Either for therapeutic purposes, or for expression of new or modified genes for research purposes, a more efficient and reliable method of delivering DNA is required and, in particular, protection of the DNA against metabolic effects is highly desirable.
A number of strategies have been proposed to achieve these aims. These include the use of liposomes [3], cationic lipids [4], which are often incorrectly referred to as xe2x80x98cationic liposomesxe2x80x99, and the use of cationic polymers such as polylysine [5] or polyornithine as DNA delivery agents.
Both oligonucleotides and DNA constructs, such as plasmids, have shown improved activity by condensation with polycations such as polylysine. In the former case chemical conjugation of oligonucleotide to the polymer is required, whereas in the latter case complexation of polymer with DNA also confers these effects. Poly-L-lysine (PLL) is believed to condense the DNA into a smaller volume, and by the excess positive charge of the complex, bind to negatively charged cell surfaces to facilitate interaction with the cell surface and uptake into the cell.
The effectiveness of polylysine-DNA complexes has been enhanced by coupling ligands to the polylysine which further facilitate binding and uptake into cells [6]. Membrane destabilising agents have been added to DNA preparations to facilitate exit of the DNA from the degradative endosomal compartments of the cell [7].
To date, few different cationic polymers have been used in this work, and the available polymers are deficient in a number of respects. Poly-L-lysine, the principal polymer presently used for this purpose, is known to be toxic above a small molecular weight [8], it does not interact stoichiometrically with DNA, and the resulting complex is unreliable, difficult to control and its properties strongly dependent on the ratio of DNA to polymer.
Ranucci et al [15] describes the synthesis of poly(amidoamine)s and suggests their use as polymeric drug carriers using covalent attachment of the drug molecule to the polymer.
Ranucci and Ferruti [12] describes hydrolyzable block copolymers containing poly(ethyleneglycol) (PEG) and poly(amidoamine) (PAA) or poly(amido-thioether-amine).
Haensler and Szoka [10] suggests that polyamidoamine cascade polymers (dendrimers prepared from branched chain poly(amidoamine)s) of a certain size may be useful in transfection of cells in culture and states that linear polycations in general are relatively cytotoxic and by themselves not very efficient, which limits their usefulness for transfection of cells in culture.
Duncan et al [16] describes poly(amidoamine)-Triton X-100 conjugates which may be useful for drug delivery.
Katayose and Kataoka [17] suggest that a PEG-poly(lysine) block copolymer as a potential DNA delivery system.
Attaching PEG chains to macromolecules and colloidal particles has been described for many biomedical products [11].
There remains a need for polymers which have improved properties for use in DNA delivery systems.
A first aspect of the invention provides a composition for delivering a biologically active polyanionic molecule, the composition comprising a linear polymer with a backbone comprising amido and tertiary amino groups arranged regularly on the said backbone and said biologically active polyanionic molecule bound to said polymer.
The said linear polymers are cationic and, depending on their nature, as discussed more fully below, the polymers have a range of physicochemical properties.
Conveniently, the said linear polymer comprises a poly(amidoamine) (PAA). Suitably, said linear polymer consists of a poly(amidoamnine). PAAs are degradable in water since they contain hydrolyzable amidic bonds in their main chain together with nucleophilic tertiary-aminic fuinctions in the position. The polymers can be synthesised from a wide variety of primary monoamines or secondary bisamines which enable full control to be exercised over the spacing and pKa of the cationic groups [9] for optimisation of the interaction with a suitable biologically active polyanionic molecule. Preferably, PAAs are water soluble, and thus facilitate the solubility of the complex. Further solubilisation of complexes can be achieved by use of the copolymers containing hydrophilic PEG chains. (Reference [9] is incorporated herein by reference.)
It is preferred if the pKa of the cationic groups is between 7 and 8. It has been found that PAA with a low pKa binds DNA less well than PAA with a high pKa. It is more preferred if the pKa of the PAA is around 8.
Thus, in a preferred embodiment, said linear polymer further comprises ethylene glycol or poly(ethyleneglycol). It is particularly preferred if the linear polymer is a poly(ethyleneglycol)-poly(amidoamine) block copolymer or ethyleneglycol-poly(amidoamine) block copolymer.
Preferably said linear polymer is a block copolymer with the structure [poly(amidoaniine)-(ethyleneglycol)y]x wherein x is from 1 to 50 and y is from 1 to 200, wherever it may occur.
Also preferably said linear polymer is a block copolymer with the structure (ethyleneglycol)y-poly(amidoamine)-(ethyleneglycol)y wherein each y is independently 1 to 200.
Suitably the linear polymer consists of or comprises a PAA which has the formula: 
or said linear polymer comprises a PAA such as with the formula: 
or (e) wherein 
are replaced by 
and, in any case, z is from 0 (or 1 as appropriate) to 70 and each R1 is independently H or a linear or branched hydrocarbon chain xe2x80x94CnH2n+1 with n=1-4 whenever it occurs; each R2 is independently a linear or branched alkylene chain xe2x80x94CnH2nxe2x80x94 with n=1-4 whenever it occurs; each R3 is independently a linear or branched hydrocarbon chain xe2x80x94CnH2n+1 with n=1-4 whenever it occurs; each R4 is independently a linear or branched alkylene chain xe2x80x94CnH2nxe2x80x94 with n=2-4 whenever it occurs; each R5 is independently a linear or branched hydrocarbon chain xe2x80x94CnH2n+1 with n=1-4; each R6 is independently a linear or branched alkylene chain xe2x80x94CnH2nxe2x80x94 with n=2-4; and wherein R7, R8, R9 and R10 are independently H or linear or branched hydrocarbon chain xe2x80x94CnH2n+1 with n=1-3 whenever they occur.
Preferably Z is from 30 to 70.
It is preferred if the Mn of the PAA is greater than 10 000; more preferably greater than 15 000.
Conveniently the linear polymer has the formula: 
wherein PAA has the formula as defined above, x is from 1 to 50 and y is from 1 to 200.
Preferably, the linear polymer has the formula 
wherein PAA has the formula as defined above and y is from 1 to 200. The preferences for the degree of polymerisation of the PAA in the PAA-PEG copolymers is the same as for the PAA polymers (ie preferably Z is from 30 to 70).
The biologically active polyanionic molecule may be any suitable such molecule but preferably said molecule comprises a regular array of negative charges.
It is preferred if the molecule comprising a regular array of negative charges is a nucleic acid or a derivative thereof.
It is less preferred if the molecule is heparin.
The nucleic acid or derivative thereof may be DNA or RNA.
The nucleic acid may be an antisense nucleic acid. The nucleic acid is conveniently an oligonucleotide such as an antisense oligonucleotide.
If the molecule is an oligonucleotide it is preferred that the PAA has a relatively low degree of polymerisation. If the molecule is a larger DNA or RNA molecule it is preferred that the PAA has a relatively high degree of polymerisation.
The nucleic acid, as discussed below, is preferably a therapeutic nucleic acid useful in gene therapy, nucleic acid vaccination, antisense therapy and the like. As discussed in more detail below, the nucleic acid may comprise natural (phosphate) phosphodiester linkages or it may comprise non-natural linkages such as those including phosphorothioates. It is preferred if the nucleic acid is DNA or a derivative thereof.
Antisense oligonucleotides are single-stranded nucleic acid, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are often termed xe2x80x9cantisensexe2x80x9d because they are complementary to the sense or coding strand of the gene. Recently, formation of a triple helix has proven possible where the oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the DNA double helix. A triple helix was formed thereby. This suggests that it is possible to synthesise sequence-specific molecules which specifically bind double-stranded DNA via recognition of major groove hydrogen binding sites.
Clearly, the sequence of the antisense nucleic acid or oligonucleotide can readily be determined by reference to the nucleotide sequence of the gene whose function is to be interfered with.
In a still further embodiment the nucleic acid delivered to a target cell encodes an antisense RNA.
An antisense RNA includes an RNA molecule which hybridises to, and interferes with the expression from a MRNA molecule encoding a protein or to another RNA molecule within the cell such as pre-mRNA or tRNA or rRNA, or hybridises to, and interferes with the expression from a gene.
Conveniently, a gene expressing an antisense RNA may be constructed by inserting a coding sequence encoding a protein adjacent a promoter in the appropriate orientation such that the RNA complementary to MRNA. Suitably, the antisense RNA blocks expression of undesirable polypeptides such as oncogenes, for example ras, bcl, src or tumour suppressor genes such as p53 and Rb.
It will be appreciated that it may be sufficient to reduce expression of the undesirable polypeptide rather than abolish the expression.
It will be further appreciated that DNA sequences suitable for expressing as antisense RNA and for designing other antisense nucleic acids may be readily derived from publicly accessible databases such as GenBank and EMBL.
Oligonucleotides are subject to being degraded or inactivated by cellular endogenous nucleases. To counter this problem, it is possible to use modified oligonucleotides, eg having altered internucleotide linkages which retain a negative charge, in which the naturally occurring phosphodiester linkages have been replaced with another linkage. For example, Agrawal et al (1988) Proc. Natl. Acad. Sci. USA 85, 7079-7083 showed increased inhibition in tissue culture of HIV-1 using oligonucleotide phosphoramidates and phosphorothioates. Agrawal et al (1989) Proc. Natl. Acad. Sci. USA 86, 7790-7794 showed inhibition of HIV-1 replication in both early-infected and chronically infected cell cultures, using nucleotide sequence-specific oligonucleotide phosphorothioates. Leither et al (1990) Proc. Natl. Acad. Sci. USA 87, 3430-3434 report inhibition in tissue culture of influenza virus replication by oligonucleotide phosphorothioates.
Oligonucleotides having artificial linkages have been shown to be resistant to degradation in vivo. For example, Shaw et al (1991) in Nucleic Acids Res. 19, 747-750, report that otherwise unmodified oligonucleotides become more resistant to nucleases in vivo when they are blocked at the 3xe2x80x2 end by certain capping structures and that uncapped oligonucleotide phosphorothioates are not degraded in vivo.
A detailed description of the H-phosphonate approach to synthesizing oligonucleos dephosphorothioates is provided in Agrawal and Tang (1990) Tetrahedron Letters 31, 7541-7544, the teachings of which are hereby incorporated herein by reference. Syntheses of oligonucleoside phosphorodithioates and phosphoramidates are known in the art. See, for example, Agrawal and Goodchild (1987) Tetrahedron Letters 28, 3539; Nielsen et al (1988) Tetrahedron Letters 29, 2911; Jager et al (1988) Biochemistry 27, 7237; Uznanski et al (1987) Tetrahedron Letters 28, 3401; Bannwarth (1988) Helv. Chim. Acta. 71, 1517; Crosstick and Vyle (1989) Tetrahedron Letters 30, 4693; Agrawal et al (1990) Proc. Natl. Acad. Sci. USA 87, 1401-1405, the teachings of which are incorporated herein by reference. Other methods for synthesis or production also are possible. In a preferred embodiment the oligonucleotide is a deoxyribonucleic acid (DNA), although ribonucleic acid (RNA) sequences may also be synthesized and applied.
The oligonucleotides useful in the invention preferably are designed to resist degradation by endogenous nucleolytic enzymes. In vivo degradation of oligonucleotides produces oligonucleotide breakdown products of reduced length. Such breakdown products are more likely to engage in non-specific hybridization and are less likely to be effective, relative to their full-length counterparts. Thus, it is desirable to use oligonucleotides that are resistant to degradation in the body and which are able to reach the targeted cells. The present oligonucleotides can be rendered more resistant to degradation in vivo by substituting one or more internal artificial internucleotide linkages for the native phosphodiester linkages, for example, by replacing phosphate with sulphur in the linkage. The synthesis of oligonucleotides having one or more of these linkages substituted for the phosphodiester internucleotide linkages is well known in the art, including synthetic pathways for producing oligonucleotides having mixed internucleotide linkages.
Oligonucleotides can be made resistant to extension by endogenous enzymes by xe2x80x9ccappingxe2x80x9d or incorporating similar groups on the 5xe2x80x2 or 3xe2x80x2 terminal nucleotides. A reagent for capping is commercially available as Amino-Link II(trademark) from Applied BioSystems Inc, Foster City, Calif. Methods for capping are described, for example, by Shaw et al (1991) Nucleic Acids Res. 19, 747-750 and Agrawal et al (1991) Proc. Natl. Acad. Sci. USA 88(17), 7595-7599, the teachings of which are hereby incorporated herein by reference.
A further method of making oligonucleotides resistant to nuclease attack is for them to be xe2x80x9cself-stabilizedxe2x80x9d as described by Tang et al (1993) Nucl. Acids Res. 21, 2729-2735 incorporated herein by reference. Self-stabilized oligonucleotides have hairpin loop structures at their 3xe2x80x2 ends, and show increased resistance to degradation by snake venom phosphodiesterase, DNA polymerase I and fetal bovine serum. The self-stabilized region of the oligonucleotide does not interfere in hybridization with complementary nucleic acids, and pharmacokinetic and stability studies in mice have shown increased in vivo persistence of self-stabilized oligonucleotides with respect to their linear counterparts.
It is preferred that the oligonucleotides contain phosphodiester linkages.
However, the polymers of the invention can aid compaction and stabilisation of the nucleic acid and, it is believed, can protect the nucleic acid from degradation.
We have found that this family of cationic polymers, and the complexes formed, have superior properties for use as DNA delivery systems in comparison with other cationic polymers previously reported for this purpose.
Complexes between the polymers as defined, and in particular polyamidoamines and copolymers thereof, and DNA are readily formed by simple mixing at the required ratio of DNA to polymer. In contrast to complexes of poly-L-lysine and DNA, which are invariably insoluble and form colloidal particles in the size range of 100 nm to several xcexcm in diameter, the PAA-DNA complexes remain soluble under some conditions.
The use of PEG containing polymers increase the range of conditions under which soluble complexes are seen. Generally PEGylation has the effect of reducing interaction with scavenger receptors and cells, so prolonging the circulation half-life and reducing immunogenic responses. In the case of complexes with DNA this is also expected to reduce the metabolism of DNA by serum enzymes. The advantages of having the PEG bound to the polymer rather than directly to the DNA is that it reduces the possibility that the hydrophilic PEG will interfere with the uptake of DNA into the cell through hydrophobic membranes and hence location to the correct intracellular compartment. The synthesis of PAA and PAA-PEG, however still allows for the further conjugation of other biologically active recognition sequences to further improve the uptake of the DNA and its transfer to the correct intracellular compartment.
A preferred embodiment is wherein the polymer further comprises a biologically active recognition signal. The said signal may aid the targeting, uptake or intracellular localisation of the composition and therefore the said biologically active polyanionic molecule.
Suitable said recognition signals include ligands for binding and endocytosis especially of DNA delivery systems such as transferrin, for example see E. Wagner, M. Cotten, R. Foisner and M. L. Bernstiel (1991) Proc. Natl. Acad. Sci. USA 88, 4255-4259; carbohydrate residues, for example galactose, or mannose residues to target to hepatocytes or macrophages respectively. (G. Ashwell and J. Harford (1982) Ann. Rev. Biochem. 51, 531-54 describes carbohydrate specific receptors of the liver and use of asialoglycoprotein receptor in gene targeting with attachment of asialo-orosomucoid to PLL-DNA constructs is described in G. Y. Wu and C. H. Wu (1988) Biochemistry 27, 887-892.); folate receptors as described in C. P. Leamon and P. S. Low (1991) Proc. Natl. Acad. Sci. USA 88, 5572-5576 and G. Citro, C. Szczylik, P. Ginobbi, G. Zupi and B. Calabretta (1994) Br. J. Cancer 69, 463-464; monoclonal antibodies, especially those selective for a cell-surface antigen; and any other ligand which will mediate endocytosis of macromolecules.
Monoclonal antibodies which will bind to many of these cell surface antigens are already known but in any case, with today""s techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens. The antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]). Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in xe2x80x9cMonoclonal Antibodies: A manual of techniquesxe2x80x9d, H Zola (CRC Press, 1988) and in xe2x80x9cMonoclonal Hybridoma Antibodies: Techniques and Applicationsxe2x80x9d, J G R Hurrell (CRC Press, 1982).
Chimaeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799).
Suitably prepared non-human antibodies can be xe2x80x9chumanizedxe2x80x9d in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies.
Suitable endosome disrupting agents such as viral fusogenic peptides and adenoviral particles have been described in J-P. Bongartz, A-M. Aubertin, P. G. Milhaud and B. Lebleu (1994) Nucleic Acids Research 22, 4681-4688 and M. Cotten, E. Wagner, K. Zatloukal, S. Phillips, D. T. Curiel, M. L. Bernstiel (1992) Proc. Natl. Acad. Sci. USA 89, 6094-6098.
All of these journal articles are incorporated herein by reference.
Complexes have been formed between polymers and short single stranded DNA with both phosphodiester and phosphorothioate backbones, and high molecular weight double stranded DNA. The complexes formed have been characterised by microcalorimetry, DNA melting profiles, gel-shift electrophoresis and photon correlation spectroscopy.
It is most preferred if the biologically active polyanionic molecule is a therapeutic molecule such as a therapeutic nucleic acid.
Therapeutic nucleic acids include any nucleic acid that it is useful to deliver to a patient, and includes nucleic acid vaccines.
Preferably the nucleic acid is suitable for gene therapy.
In one embodiment, the nucleic acid encodes a molecule having a directly or indirectly cytotoxic function. By xe2x80x9cdirectly or indirectlyxe2x80x9d cytotoxic, we mean that the molecule encoded by the gene may itself be toxic (for example ricin; tumour necrosis factor; interleukin-2; interferon-gamma; ribonuclease; deoxyribonuclease; Pseudomonas exotoxin A) or it may be metabolised to form a toxic product, or it may act on something else to form a toxic product. The sequence of ricin cDNA is disclosed in Lamb et al (1985) Eur. J. Biochem. 148, 265-270 incorporated herein by reference.
For example, it would be desirable to deliver to cancer cells within a patient a nucleic acid encoding an enzyme using the compositions of the invention, the enzyme being one that converts a relatively non-toxic prodrug to a toxic drug. The enzyme cytosine deaminase converts 5-fluorocytosine (5FC) to 5-fluorouracil (5FU) (Mullen et al (1922) PNAS 89, 33); the herpes simplex enzyme thymidine kinase sensitises cells to treatment with the antiviral agent ganciclovir (GCV) or aciclovir (Moolten (1986) Cancer Res. 46, 5276; Ezzedine et al (1991) New Biol 3, 608). The cytosine deaminase of any organism, for example E. coli or Saccharonzyces cerevisiae, may be used.
Thus, in one embodiment of the invention, the nucleic acid encodes a cytosine deaminase and the patient is concomitantly given 5FC. By xe2x80x9cconcomitantlyxe2x80x9d, we mean that the 5FC is administered at such a time, in relation to the transformation of the tumour cells, that 5FC is converted into 5FU in the target cells by the cytosine deaminase expressed from the said gene. A dosage of approximately 0.001 to 100.0 mg 5FC/kg body weight/day, preferably 0.1 to 10.0 mg/kg/day is suitable.
Components, such as 5FC, which are converted from a relatively non-toxic form into a cytotoxic form by the action of an enzyme are termed xe2x80x9cpro-drugsxe2x80x9d.
In a further embodiment the nucleic acid delivered to the target cell is or encodes a ribozyme capable of cleaving targeted RNA or DNA. The targeted RNA or DNA to be cleaved may be RNA or DNA which is essential to the function of the cell and cleavage thereof results in cell death or the RNA or DNA to be cleaved may be RNA or DNA which encodes an undesirable protein, for example an oncogene product, and cleavage of this RNA or DNA m ay prevent the cell from becoming cancerous.
Ribozymes which may be encoded in the genomes of the viruses or virus-like particles herein disclosed are described in Cech and Herschlag xe2x80x9cSite-specific cleavage of single stranded DNAxe2x80x9d U.S. Pat. No. 5,180,818; Altaa et al xe2x80x9cCleavage of targeted RNA by RNAse Pxe2x80x9d U.S. Pat. No. 5,168,053, Cantin et al xe2x80x9cRibozyme cleavage of HW-1 RNAxe2x80x9d U.S. Pat. No. 5,149,796; Cech et al xe2x80x9cRNA ribozyme restriction endoribonucleases and methodsxe2x80x9d, U.S. Pat. No. 5,116,742; Been et al xe2x80x9cRNA ribozyme polymerases, dephosphorylases, restriction endonucleases and methods, U.S. Pat. No. 5,093,246; and Been et al xe2x80x9cRNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods; cleaves single-stranded RNA at specific site by transesterificationxe2x80x9d, U.S. Pat. No. 4,987,071, all incorporated herein by reference.
In another embodiment of the invention, the nucleic acid replaces the function of a defective gene in the target cell.
There are several thousand inherited genetic diseases of mammals, including humans, that are caused by defective genes. Examples of such genetic diseases include cystic fibrosis, where there is known to be a mutation in the CFTR gene; Duchenne muscular dystrophy, where there is known to be a mutation in the dystrophin gene; sickle cell disease, where there is known to be a mutation in the HbA gene. Many types of cancer are caused by defective genes, especially protooncogenes, and tumour-suppressor genes that have undergone mutation.
The following table shows current targets for gene therapy.
This list indicates the principal current targets for gene therapy. Many of the diseases listed can be caused by defects in more than one gene; the gene defect listed is the defect targeted by current research.
Thus, it is preferred that the composition of the invention, which may be useful in the treatment of cystic fibrosis, contains a functional CFTR gene to replace the function of the defective CFTR gene. Similarly, it is preferred that the virus or virus-like particle of the invention, which may be useful in the treatment of cancer, contains a functional protooncogene, or tumour-suppressor gene to replace the function of the defective protooncogene or tumour-suppressor gene.
Examples of protooncogenes are ras, src, bcl and so on; examples of tumour-suppressor genes are p53 and Rb.
The nucleic acid may contain introns, or it may be a gene or a fragment thereof, or cDNA, or fragment thereof.
Nucleic acids suitable for use in vaccines of the present invention include those described in Volume 12(16) of Vaccine which is a special conference issue of the WHO meeting on nucleic and vaccines, and is incorporated herein by reference. Nucleic acid vaccines for tuberculosis, influenza, hepatitis B, Leishmaniasis and HIV have been considered.
The nucleic acid, especially DNA, which is bound to the polymer in the composition of the invention may be any suitable size. Preferably the nucleic acid is from 10 to 10 million bases or base pairs. Suitably oligonucleotides are from 10 bases to 200 bases, more suitably 10 bases to 100 bases.
Conveniently RNA and DNA molecules are from 100 to 1 million bases or base pairs.
More preferably the nucleic acid is from 20 to 1 million bases or base pairs, still more preferably the nucleic acid is from 1000 to 500,000 bases or base pairs and most preferably the nucleic acid is from 5000 to 150,000 bases or base pairs.
The nucleic acid may conveniently be plasmid DNA whether supercoiled, open circle or linearised plasmid DNA.
It is believed that the nucleic acid binds to the polymer non-covalently.
A second aspect of the invention provides a composition according to the first aspect of the invention for use in medicine.
A third aspect of the invention provides a composition according to the first aspect of the invention in the manufacture of a medicament for treatment of a disease.
A fourth aspect of the invention provides a pharmaceutical composition comprising a composition according to the first aspect of the invention and a pharmaceutically effective carrier.
The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (composition of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.
It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
A fifth aspect of the invention provides a method of making a composition according to the first aspect of the invention comprising contacting said biologically active polyanionic molecule with said linear polymer.
Preferably, the biologically active polyanionic molecule and the said linear polymer are simply mixed together, preferably in solution, more preferably in aqueous solution. They may be mixed together quickly or slowly. Preferably, the biologically active polyanionic molecule is a nucleic acid, more preferably DNA.
When nucleic acid is used, it is convenient to mix the nucleic acid and polymer in a high salt solution and dialyse against water. This method is particularly preferred for large nucleic acid molecules such as those  greater than 1 kb. It is also convenient to heat the mixture of polymer and nucleic acid and to cool the mixture slowly. Preferably, the mixture is a solution, more preferably an aqueous solution.
A sixth aspect of the invention provides a method of delivering a biologically active polyanionic molecule to a host, the method comprising administering to said host an effective amount of a composition according to the first aspect of the invention.
The host is suitably a patient to be treated with the biologically active polyanionic molecule.
The host may be, for example, a cell in culture in vitro or it may be an experimental animal.
Preferably, the biologically active polyanionic molecule is a nucleic acid, more preferably DNA.
When the host is a cell in culture the method can be used to transfect or transform the cell with the nucleic acid, preferably DNA.
A seventh aspect of the invention provides a method of delivering a biologically active polyanionic molecule to a cell in an environment, the method comprising administering to said environment a composition as defined in the first aspect of the environment.
The environment may be a patient to be treated or an experimental animal to be treated or a culture medium containing cells.
Preferably the environment is a culture medium containing cells. When the biologically active polyanionic molecule is a nucleic acid the cells may be transfected or transformed using this method by administering a suitable composition to the culture medium.
By xe2x80x9ccellsxe2x80x9d we include both prokaryotic and eukaryotic cells. Thus, the cells include cells of bacteria, yeast, fungi, plants, vertebrates (such as mammalian cells) and invertebrates (such as insect cells).
Preferably the nucleic acid to be delivered is DNA.
Preferably in this method the said composition is contacted with said cell.
Preferably for the methods described in the fifth, sixth and seventh aspects of the invention the biologically active polyanionic molecule is a therapeutic molecule and more preferably the therapeutic molecule comprises a nucleic acid.
An eighth aspect of the invention provides a method of treating, preventing or ameliorating a disease in a multicellular organism which multicellular organism benefits from the administration of a biologically active polyanionic molecule, the method comprising administering to the patient a composition as defined in the first aspect of the invention.
Preferably the biologically active polyanionic molecule comprises a therapeutic nucleic acid or derivative thereof.
The multicellular organism may be an animal or human or a plant, preferably an animal especially a mammal and more preferably a human.
The animal or human is therefore a patient.
The aforementioned compositions may be administered to the plant in any suitable way.
The aforementioned compositions of the invention or a formulation thereof may be administered to the patient by any conventional method including oral and parenteral (eg subcutaneous or intramuscular) injection. The treatment may consist of a single dose or a plurality of doses over a period of time.
Whilst it is possible for a composition of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline or buffered solutions preferably wherein the buffer buffers in a physiological pH range which will be sterile and pyrogen free.
A ninth aspect of the invention provides a polymer with the formula (ethylene glycol)y-poly(amidoamine)-(ethyleneglycol)y wherein each y is independently from 1 to 200.
Preferably the compound has the formula 
wherein PAA is a poly(amidoamine) as defined above and each y is independently from 1 to 200.
This polymer can be used as the polymer in all previous aspects of the invention and it may also be useful in binding drugs generally for delivery to a patient to be treated.